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Increased canonical Wnt activity in Lrrk2 knockout cells. A, B) Cells were transfected with TK-renilla and TOPflash or FOPflash in the presence of A) FLAG-DVl1 or C) GFP-FZD5 and HA-LRP6, or appropriate vector controls. A) 1-way ANOVA (n = 9; F = 146.199, p

Association of mycLRRK2 and FLAG-β-catenin in HEK293 cells 24 h post transfection, cells expressing myc-tagged LRRK2 and FLAG-tagged β-catenin were treated with MG132 for 1 h to allow β-catenin accumulation. The cells were subsequently fixed and stained with antibodies for A) myc (green) and FLAG (red), or B) myc (green) and β-catenin (red). DAPI staining (blue) was also performed to show cell nuclei. The right-hand panel shows an overlay of all channels – note the considerable co-localisation between LRRK2 and β-catenin in the cytoplasm. (PDF 102 kb)

Effects of loss of Lrrk2 on tibial cortical Zmax and Zmin. Images in Figures A and B show i) values for Zmax and Zmin, predicted resistance to fracture along the shortest and longest cross-sectional axes, respectively in female wild-type (WT) and Lrrk2 knockout (KO) mice; together with ii) the same data expressed as a graphical heat map along the tibial length; and iii) the points at which differences between genotypes for these parameters become statistically significant. (blue = n/s, yellow, p

Effects of loss of Lrrk2 on tibial cortical Zmax and Zmin. Images in Figures A and B show i) values for Zmax and Zmin, predicted resistance to fracture along the shortest and longest cross-sectional axes, respectively in female wild-type (WT) and Lrrk2 knockout (KO) mice; together with ii) the same data expressed as a graphical heat map along the tibial length; and iii) the points at which differences between genotypes for these parameters become statistically significant. (blue = n/s, yellow, p

Effects of loss of Lrrk2 on tibial cortical Cross Sectional Area, Mean Cortical Thickness, and Ellipticity. Images in Figures A, B and C show i) values for cross-sectional area, mean cortical thickness and ellipticity, respectively in female wild-type (WT) and Lrrk2 knockout (KO) mice; together with ii) the same data expressed as a graphical heat map along the tibial length; and iii) the points at which differences between genotypes for these parameters become statistically significant. (blue = n/s, yellow, p

LRRK2 interacts with β-catenin and represses β-catenin-driven TOPflash activity. A) HEK293 cells were co-transfected with FLAG-tagged β-catenin (lanes 1 and 4), myc-tagged LRRK2 (lanes 2 and 5), or myc-tagged LRRK2 and FLAG-tagged β-catenin (lanes 3 and 6) for 24 h prior to lysis. Lysates were immunoprecipitated with anti-FLAG antibodies and bound protein was resolved by Western blot (lanes 4–6), with the original lysates run alongside to confirm expression of transfected protein (lanes 1–3). Myc-tagged LRRK2 can clearly be seen in anti-FLAG immunoprecipitates from co-transfected cells (lane 6, upper panel). Full images of all blots are also shown. B) HEK293 cells were transfected with the TOPflash or FOPflash reporter plasmids with the indicated combinations of mycLRRK2, FLAG-β-catenin or appropriate control vectors. After 24 h lysates were taken and luciferase activity measured. 1-way ANOVA of TOPflash values revealed a significant effect of transfection on canonical Wnt activity (n = 9, F = 44.893, p

Entire images of Western Blots shown in Fig. 4D and E and total β-catenin levels in aged female mouse brains A) Full blot from the image shown in Fig. 4d, plus a reprobe of the same membrane for calnexin to show that pulldowns are clean and this protein is only present in cell lysates. B) Western blotting shows no discernible differences between levels of total β-catenin in brains from aged female mice. C) Full blot from the image shown in Fig. 4e. (PDF 102 kb)

LRRK2 interacts with β-catenin and represses β-catenin-driven TOPflash activity. A) HEK293 cells were co-transfected with FLAG-tagged β-catenin (lanes 1 and 4), myc-tagged LRRK2 (lanes 2 and 5), or myc-tagged LRRK2 and FLAG-tagged β-catenin (lanes 3 and 6) for 24 h prior to lysis. Lysates were immunoprecipitated with anti-FLAG antibodies and bound protein was resolved by Western blot (lanes 4–6), with the original lysates run alongside to confirm expression of transfected protein (lanes 1–3). Myc-tagged LRRK2 can clearly be seen in anti-FLAG immunoprecipitates from co-transfected cells (lane 6, upper panel). Full images of all blots are also shown. B) HEK293 cells were transfected with the TOPflash or FOPflash reporter plasmids with the indicated combinations of mycLRRK2, FLAG-β-catenin or appropriate control vectors. After 24 h lysates were taken and luciferase activity measured. 1-way ANOVA of TOPflash values revealed a significant effect of transfection on canonical Wnt activity (n = 9, F = 44.893, p

Effects of loss of Lrrk2 on tibial cortical Cross Sectional Area, Mean Cortical Thickness, and Ellipticity. Images in Figures A, B and C show i) values for cross-sectional area, mean cortical thickness and ellipticity, respectively in female wild-type (WT) and Lrrk2 knockout (KO) mice; together with ii) the same data expressed as a graphical heat map along the tibial length; and iii) the points at which differences between genotypes for these parameters become statistically significant. (blue = n/s, yellow, p

Co-localisation between mycLRRK2 and HA-AXIN1, and mycLRRK2 and HA-GSK3β in HEK293 cells. A) Recruitment of mycLRRK2 (green) into polymers formed by HA-AXIN1 (red). Magnified images of a selected region are included. B) shows cytoplasmic co-localisation between mycLRRK2 (green) and HA-GSK3β (red), also with magnified images included. Note that in both experiments, counterstaining with phalloidin (blue) and DAPI (grey) were performed to visualise filamentous actin and chromosomal DNA respectively. The scale bar = 10 μm. (PDF 92 kb)

Entire images of Western Blots shown in Fig. 4a. (PDF 70 kb)

Co-localisation between mycLRRK2 and HA-AXIN1, and mycLRRK2 and HA-GSK3β in HEK293 cells. A) Recruitment of mycLRRK2 (green) into polymers formed by HA-AXIN1 (red). Magnified images of a selected region are included. B) shows cytoplasmic co-localisation between mycLRRK2 (green) and HA-GSK3β (red), also with magnified images included. Note that in both experiments, counterstaining with phalloidin (blue) and DAPI (grey) were performed to visualise filamentous actin and chromosomal DNA respectively. The scale bar = 10 μm. (PDF 92 kb)

Entire images of Western Blots shown in Fig. 4a. (PDF 70 kb)

Association of mycLRRK2 and FLAG-β-catenin in HEK293 cells 24 h post transfection, cells expressing myc-tagged LRRK2 and FLAG-tagged β-catenin were treated with MG132 for 1 h to allow β-catenin accumulation. The cells were subsequently fixed and stained with antibodies for A) myc (green) and FLAG (red), or B) myc (green) and β-catenin (red). DAPI staining (blue) was also performed to show cell nuclei. The right-hand panel shows an overlay of all channels – note the considerable co-localisation between LRRK2 and β-catenin in the cytoplasm. (PDF 102 kb)

Entire images of Western Blots shown in Fig. 5b. (PDF 80 kb)

Increased canonical Wnt activity in Lrrk2 knockout cells. A, B) Cells were transfected with TK-renilla and TOPflash or FOPflash in the presence of A) FLAG-DVl1 or C) GFP-FZD5 and HA-LRP6, or appropriate vector controls. A) 1-way ANOVA (n = 9; F = 146.199, p

Kinase-dead but not GTP-non-binding mutations weaken basal Wnt signalling. Lrrk2 knockout (KO) cells were transfected with TOPflash or FOPflash plus wild-type LRRK2 or the indicated LRRK2 mutant. 1-way ANOVA (n = 12–15, F = 7.449, p

Entire images of Western Blots shown in Fig. 4D and E and total β-catenin levels in aged female mouse brains A) Full blot from the image shown in Fig. 4d, plus a reprobe of the same membrane for calnexin to show that pulldowns are clean and this protein is only present in cell lysates. B) Western blotting shows no discernible differences between levels of total β-catenin in brains from aged female mice. C) Full blot from the image shown in Fig. 4e. (PDF 102 kb)